Tan, ElaineTorrelavega, Leda DaleVillahermosa, Mheira
Dr. Voltaire Organo
Tubulins Make Up Microtubules Microtubules perform
several functions in biological systems: Cell division Nerve cell
differentiation Transport inside the
cells
Tubulin undergoes Posttranslational Modifications
Since posttranslational modifications take place, we can differentiate between different populations of microtubules.
Choice of Tubulin Subunit as “Differentiator”
To do so, we need to investigate on the number of Glu-units which can vary from 1 to 20.
The Need to Know the Oligo-Glu Length Monoclonal antibodies:
Can detect the presence or absence of Glu-units But insensitive to length of modification
Clearly, the need to synthesise antibodies that are sensitive to length modification of tubulins is a must.
THIS STUDY ADDRESSES THE PROBLEM.
Objective
General Objective:
To generate specific antibodies that can discriminate between the different lengths of oligo-Glu modifications
Specific Objective:
To synthesise peptides with corresponding defined side chains
Strategy # 1
1. Incorporate main chain Glu with selectively cleavable allyl side-chain protection
2. Selectively deprotect
3. Activate carboxylic group on the resin
4. Couple H-Glu(tBu)-Oallyl with further side-chain elongation after allyl cleavage
Not reliable. Only one to two side-chain Glu residues could be introduced.
Strategy # 2
1. Preassemble suitably protected building blocks with required side-chain length
2. Utilize their assembly in the main chain
The Target PeptidesTARGET PEPTIDE # 1:
CYEEVGVDSVEGEG-E(Ex)-EEGEEY
TARGET PEPTIDE # 2:
CQDATADEQG-E(Ex)-FEEEGEDEA
These target peptides are model peptide sequences.
Target 1: Mammalian alpha-1-tubulin
Target 2: Mammalian beta-1-tubulin
Step 1. Resin Loading
But first, what is a resin? Solid supports in which reactions can
occur Insoluble polymer Must be a well-solvated system Physically stable, permit rapid filtration Inert to reagents and solvents Must swell just right
Too little swelling: reagents will not penetrate
Too much swelling: may not fit wells of automated synthesizers
Chlorotrityl Polystyrene Resin Highly-solvated Hydrophobic beads are solvated by
nonpolar solvents such as dichloromethane
Fmoc: 9-fluorenylmethyloxycarbamoyl chlorideServes as protecting group for amines.
Cl- serves as leaving group.Lone pair of Nitrogen amine attacks
carbonyl C, displacing Cl-.
(the protected amino acid)
Protection of Amino Acid Unit
At this point, the Fmoc-protected unit is acid-stable.
Diisopropylethylamine Tertiary amine Used as a base N atom: shielded by 2 isopropyl groups & ethyl group, only 1 proton can
easily fit Readily attacks unprotected –OH group
Coupling of Protected Glu Unit to Resin
Dichloromethane Hydrophobic solvent
At this point, the Fmoc-protected unit has been coupled to the resin.
Deprotection of The Fmoc-protected Unit
Piperidine Heterocyclic amine Serves as a base that cleaves off the Fmoc group Deprotects N-terminus, freeing it for another coupling reaction(Reaction mechanism shown on next slide.)
Dimethylformamide Hydrophilic Common solvent Washes away the coupling reagents previously usedMust be kept in the dark. May undergo photolysis to form CO
and dimethylamine.
How Deprotection Occurs
Dibenzofulvene AdductCarbon Dioxide
Deprotected Glu Unit
Progress of deprotection can be followed by quantifying the adduct.
The deprotected amino group of the Glu unit is neutral.
Minimizes the use of other reagents.
Prevents aggregation of peptides during neutralization of the amino group and coupling of the next amino acid.
Addition of Glu-unit was performed (x-1) more times.
Oligo – Glu unit
Step 2. Assembly of Precursors
• Has 2 protecting groups
- Fmoc
- Allyl
-COOH Activation• The carboxyl groups must first be activated in order for coupling to occur.
• There are usually two types of activators – carbodiimides (too reactive, cause racemization) and triazolols (reactivity just right).
TBTU converts –COOH group into a more reactive -COOR group.
Peptide Coupling
Where the solvent used is DMF.
Why do we need to activate?
• C-terminus: site where amino acid monomers are added
• increase electrophilicity of carboxylate group
• make oxygen a better leaving group
At this point, the peptide has been coupled. Cleavage from the resin can now take place.
Cleavage from Resin
Trifluoroacetic Acid
• Strong carboxylic acid
• Volatile
• Electronegative trifluoromethyl group
• Cleaves peptide from resin
At this point, the peptide has been cleaved from the resin.
Step 3. Formation of t-Butyl Esters
Upon cleavage using TFA, you get a free carboxylic function.
Free –OH can be esterified.
Why do we need to esterify?
We need to esterify to get a good leaving group for the incorporation of the Oligo-Glu building block into the peptide sequences.
Cyclohexane
• Unreactive
• Non-polar
• Hydrophobic
• Serves as a solvent
Acetic Acid
• Serves as a Lewis acid
• activates C=C bond (???)
• upon activation, N nucleophile can attack
• trichloroacetimidate serves as a leaving group
The problem with the use of acetic acid:
• Acetic acid can react with t-butyl trichloroacetimidate (How???)
The solution:
• Have a tenfold-excess of t-butyl trichloroacetimidate
At this point, we have formed the t-butyl ester. The next step is to deprotect by cleaving the allyl group.
Step 4. Cleavage of the Allyl Group
Tetrakis Palladium (0)
• Serves as a catalyst
• Process begins by oxidative addition to Pd(0) center
1,3-dimethylbarbituric acid
•Cleaves allyl and deprotects, giving –OH
Why use 1,3-dimethylbarbituric acid instead of TFA?
• TFA is too strong, it can cause degradation.
At this point, we have finally formed the oligo-Glu building block.
Let’s look at the yields of what we’ve done so far. Assembly of Precursors: 95% - 98% Formation of t-Butyl Esters: 80% - 90% Cleavage of the Allyl Group: 80% - 90%
Step 5. Incorporation of the Oligo-Glu Building Blocks into the Peptide SequencesTARGET PEPTIDE # 1:
CYEEVGVDSVEGEG-E(Ex)-EEGEEY
At this point, what’s left Is to remove the protecting groups and cleave the peptide from the resin.
Final Removal of Protecting Groups
Triisopropylsilane (TIPS)
• Serves as a scavenger
• Removes the tBU protecting groups
• (Look for mechanism.)
Cleavage from Resin
This is accomplished by the use of TFA.
Finally, we have formed the first target peptide.
TARGET PEPTIDE # 2:
CQDATADEQG-E(Ex)-FEEEGEDEA
The assembling of the target peptide # 2 is similar to that of target peptide # 1.
Orthogonality of the Fmoc method proves to be an advantage.
An orthogonal process means that a final product occurs by 2 independent mechanisms.
• Deprotection of alpha-amino group: piperidine/DMF, TFA
• Final cleavage from resin: TIPS and TFA
The yields obtained for the two target peptides were not given.
The description given was “good yield and purity”.
Conclusion
The Fmoc-method successfully synthesized the target peptides to be used as an antibody in investigating on oligoglutamylation of tubulin upon undergoing posttranslational modifications.
Thank you!
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